Millions of people in the United States alone are afflicted with painful inflammatory or degenerative arthritis which limits normal joint function and results in loss of quality of life. The primary cause of degenerative arthritis is breakdown of the cartilage matrix. Cartilage is a smooth, flexible connective tissue covering the ends of a joint which functions to cushion the bone and allow the joint to move easily without pain. Loss of joint articular cartilage due to traumatic injury or disease ultimately results in joint stiffening caused by “bone on bone movement” and painful exposure of nerve endings in subchondral bone.
In mammals, cartilage contributes to the structure of several organs and systems like the articular surface of joints and other joint-associated structures, including the ear, the nose, the larynx, the trachea, the bronchi, structures of the heart valves, etc. There are different types of cartilage in mammals: fibro-cartilage, elastic cartilage and hyaline cartilage. Fibro-cartilage contains an abundance of type I collagen and is found in the intervertebral disks and ligaments. Elastic cartilage contains elastin fibrils and is found in the pinna of the ear and in the epiglottis. Hyaline cartilage, a semi-transparent and clear cartilage tissue found in the cartilagenous walls of the trachea and bronchia, the costal cartilage and growth plate, as well as in cartilage of the nose, larynx and diarthroidal joints, contains neither type I collagen nor elastin. Hyaline cartilage having a distinctive combination of cartilage-specific collagens (types II, VI, IX, and XI) and aggregating proteoglycans (aggrecan) that give it the unique ability to withstand compressive forces is called articular cartilage.
Damage to articular cartilage results in lesions of the joint surface, and progressive degeneration of these lesions often leads to symptomatic joint pain, disability and reduced or disturbed functionality. Joint surface defects can be the result of various aetiologies, including inflammatory processes, neoplasias, post-traumatic and degenerative events, etc. Adult articular cartilage has a major shortcoming: unlike most tissues, it cannot repair itself. Lack of a blood supply in large part restricts the tissue's ability to recruit chondroprogenitor cells that can act to repair articular cartilage defects. Consequently, articular cartilage defects that have progressed to advanced degenerative disease require total joint arthroplasty to eliminate pain and to restore normal joint function.
Tissue-engineered growth of articular cartilage represents a biologic solution which may delay or reduce the need for metal- and polymer-based materials currently used in total joint arthroplasty. Several biologic approaches have attempted to repair or regenerate articular cartilage that is damaged by trauma or disease (Hunziker, 2003). The majority of these approaches combine cell-based therapy with biodegradable polymers to create a three-dimensional construct that can be transplanted into the knee. However, these experimental therapies have not produced long-lasting repair of hyaline cartilage (Buckwatler et al., 1990; Hunziker, 2002).
One technique that has gained FDA approval for cartilage repair is Autologous Chondrocyte Implantation (Carticel, Genzyme Surgery). In this procedure, a small tissue biopsy obtained from the patient's joint articular cartilage is taken to the lab where chondrocytes (cartilage cells) are isolated and expanded ex vivo for subsequent re-implantation into the patient in a second surgical procedure. A key limitation of this method is the relatively small number of donor cells that can be obtained at biopsy, and chondrocytes derived from adult articular cartilage appear to have a limited ability to produce cartilage matrix after expansion.
A successful tissue engineered approach to cartilage repair must make use of cells that can be expanded in a scalable process that is both efficient and reproducible and which retains the ability of the expanded chondrocytes to synthesize functional cartilage for use in transplantation. Currently, the most widely used technique for chondrocyte expansion is monolayer culture (U.S. Pat. No. 4,356,261). However, chondrocytes grown in monolayer culture using serum-containing medium undergo a process of dedifferentiation in which chondrocytes lose their spherical shape and acquire an elongated fibroblastic morphology. Biochemical changes associated with loss of native chondrocyte shape include arrested synthesis of cartilage-specific collagens and proteoglycans, subsequent initiation of type I and III collagen synthesis and increased synthesis of small non-aggregating proteoglycans.
Loss of chondrocyte phenotype during serial expansion in vitro poses a key limitation to the commercialization of orthobiologic approaches to articular cartilage repair. To counter dedifferentiation, chondrocytes traditionally have been suspended in three-dimensional environments such as hydrogels, e.g., agarose (Benya and Shafer, 1982) or alginate (Hauselmann et al., 1994 and 1996), pellet culture (Mackay et al., 1998; Jakob et al., 2001; Barbero et al., 2004), or three-dimensional scaffolds (Vacanti et al., 1998). Chondrocytes are reported to better retain their native rounded morphologic appearance and to synthesize macromolecules characteristic of hyaline cartilage when maintained in three-dimensional suspension culture after expansion. However, many of such cultured chondrocytes still produce type I collagen and small proteoglycans, indicating an “incompletely” restored cartilage phenotype. Furthermore, the potential for carry over of residual materials derived from the three-dimensional hydrogels can complicate the regulatory path. Alginate, for example, is reported to induce inflammation and may be cytotoxic when used in vivo.
A logical approach to retain chondrocyte phenotype during in vitro expansion would be to recapitulate the in vivo microenvironment to which chondrocytes are naturally exposed during embryonic development. Therefore, matrices such as type II and VI collagen or aggregating proteoglycans may serve as excellent substrata for chondrocyte expansion and growth, particularly in the absence of serum-derived factors.
During embryonic development, condensation and proliferation of mesenchymal progenitor cells forms the cartilage anlagen through a process known as chondrogenesis. Further differentiation of the cartilage template results in formation of joint articular cartilage and bone. Many factors are believed to play a critical role in chondrogenesis, including the extracellular matrix, growth and differentiation factors, their antagonists as well as specific cell surface membrane receptors including, N-cadherin, bone morphogenetic protein receptor type 1A (BMPR-1A) and bone morphogenetic protein receptor type 1B (BMPR-1B).
An important component of the extracellular matrix, hyaluronic acid (HA) plays a critical role in cartilage development and in the maintenance of tissue homeostasis. HA is a non-sulfated, linear glycosaminoglycan of the extracellular matrix consisting of repeating units of(β, 1-4) D-glucuronic acid-(β1-3)—N-acetyl-D-glucosamine (Laurent, 1970). HA is ubiquitously distributed in body tissues and has been shown to play an important role in a number of biological processes including embryonic development, wound healing and tumor growth by providing a provisional matrix that supports cellular migration, adherence, proliferation and differentiation (Laurent and Fraser, 1992). In its native state, HA exists as a high molecular weight polymer, usually in excess of 1×106 daltons. However, during morphogenesis, inflammation and tissue repair reduced molecular weight forms are generated by proteolytic cleavage. Hyaluronic acid of intermediate molecular weight (200,000 to 400,000) is reported to promote differentiation of chondrogenic progenitor cells (Kujawa et al., 1986 A and 1986B), whereas HA of reduced MW promotes angiogenesis (West et al., 1985). Such findings have led to the commercial development of HA-based scaffolds for tissue engineered growth of cartilage and bone, as a means of regenerating tissues that have been destroyed by trauma or disease (Campoccia et al., 1998 U.S. Pat. Nos. 6,251,876; 5,676,964; 5,658, 582).